Field of the Invention
The invention set forth herein relates to genetic engineering and recombinant cells useful in producing terpenes and terpenoids by increasing production or accumulation or both of isoprenoid precursors thereof. The invention provides recombinant cells and methods for using such cells having reduced enzymatic activity for farnesyl diphosphate synthase, geranyl diphosphate synthase or an enzyme having both farnesyl diphosphate synthase and geranyl diphosphate synthase activities, and methods of use thereof. The recombinant cells provided by the invention generally have higher metabolic flux through the mevalonate biochemical pathway, and can also comprise additional recombinant expression constructs encoding enzymes useful for increasing products of the mevalonate pathway, particularly isoprenoids.
Background of the Related Art
Terpenes and the related terpenoids comprise a large class of biologically derived organic molecules. Terpenes and terpenoids are derived from five-carbon isoprene units and are accordingly also referred to as isoprenoids. They are produced from isoprenoid pyrophosphates which are organic molecules that serve as precursors in the biosynthesis of a number of biologically and commercially important molecules.
Terpenoids can be found in all classes of living organisms, and comprises the largest group of natural products. Plant terpenoids are used extensively for their aromatic qualities and play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastic, and other pharmaceutical functions. Terpenoids contribute to the scent of eucalyptus, the flavors of cinnamon, cloves, and ginger, and the color of yellow flowers. Well-known terpenoids include citral, menthol, camphor, Salvinorin A in the plant Salvia divinorum, and cannabinoids.
While the biosynthetic steps leading from isopentenylpyrophosphate (IPP) and/or dimethylallylpyrophosphate (DMAPP) to terpenoids are universal, two different pathways leading to IPP and DMAPP exist—the mevalonic acid pathway and the non-mevalonic, 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway. The mevalonate pathway is responsible for the production of isoprenoid-derived molecules in numerous organisms. Many isoprenoid molecules have high commercial value and production of some of these molecules in genetically engineered hosts rather than in the natural host is highly desirable for economical and sustainability reasons.
The part of the mevalonate pathway that generates the basic C5 isoprenoid pyrophosphates, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) comprises seven enzymatic steps. The seven S. cerevisiae genes involved in these steps are (in consecutive order in the pathway): ERG10, ERG13, HMGR, ERG12, ERG8, ERG19 and ID11. IPP and DMAPP are the isoprene units that form the basis for synthesis of higher order isoprenoid pyrophosphate precursors containing any number of isoprene units between two and ten. The most important ones are geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP).